miércoles, 28 de abril de 2010

SCIENTIFIC AMERICAN,Apr 23, 2010 05:35 PM in Health & MedicineIs male circumcision a humanitarian act?By Jesse BeringSo there’s this fellow—an inquisitive sort, even if not particularly bright—whom one day is asked by his ogress of a wife to drive to the store to buy a ham. Obediently, he does so, finds an impressive specimen of meat at the store, returns home and, grinning widely, places it proudly on the kitchen table before the woman. “You idiot!” screams the boorish wife. “Why didn’t you have the butcher cut off the end?” Now, our man might be dim-witted, but he’s not without a certain practical mindedness. So he asks his wife, meekly, why the end must be cut off. “Ach,” she grumbles, heaving the ham onto the counter where she begins fussily carving off the end. “Such a stupid question. That’s just the way it’s done. Mother did it and her mother before her and her mother before her.” There’s a rather puzzling hesitation in his wife’s answer that intrigues the man, however. So, still perplexed, he phones his mother-in-law—who apparently is as pleasant as his wife is unpleasant—and asks the old lady why she cuts off the ends of her hams. “You know,” says the woman, “I once posed this very question to my own grandmother. And do you know what she told me? Her old baking tray was so small that she had to cut off the end of the ham to fit it inside!”There are many different versions of this parable of the severed ham, but the moral of the story is this: many of today’s rituals—even sacred ones held by uncompromisingly stern authority figures—are just empty repetitions of a utilitarian past. In considering male circumcision, this tale of a needlessly removed hunk of ham doesn’t require a terribly huge stretch of our imaginations to apply. The surgical removing of foreskin from a neonate’s penis is seen by many critics as an outdated, cruel and unnecessary procedure that—although it may once have had some practical purpose in times past—is now done primarily out of blind habit and unquestioning obedience to “because I said so” authority figures. Although male circumcision is rare in Europe except for Jewish and Islamic subpopulations for which the procedure is a core part of religious group identity, about 70 percent of U.S. males—regardless of their religion—are circumcised. It’s embarrassing to admit, but growing up in Middle America, I didn’t even know what a nonmutilated penis looked like until the advent of the Internet. (Then again, I was also 14 before I realized that condoms weren’t, in fact, what old people in Florida lived in.) According to a 2003 report, the only meaningful predictor of whether parents will opt to get their infant son circumcised is whether the father’s own penis is circumcised—there’s a positive correlation, in case you’re wondering. Cut off the end of the ham—er, penis—because that’s what my daddy did and his daddy before him.But much as I may want to join the cause to save the prepuces, the anti-circumcision stance may not be as humanitarian as it appears. Objection on the grounds that male circumcision is a somewhat bizarre, bloody and frightening ritual was probably very reasonable throughout much of modern history. But this ancient practice, which dates back at least to the Neolithic period, just happens to have important health implications today that are completely unrelated to the hollow rituals of our forebears’ foreskin removal. This strange procedure of lopping off the ends of penises may well have persevered over the eons on the shakiest grounds of justification, grounds that invoked religious, cultural or aesthetic reasons. But, ironically, it may now finally be playing a serious—even heroic—role in staving off a much more crippling problem:

According to researchers writing in a 2009 issue of AIDS Patient Care and STDs, the prophylactic effect of male circumcision is owed to the following physical facts:There are high densities of HIV target cells in the inner mucosal surface of the human foreskin … These HIV target cells lie beneath a protective layer of keratin, which is absent on the inner surface of the prepuce. By removing all or part of the foreskin, circumcision reduces both the number and susceptibility of target cells to HIV infection.Since 2007, several randomized clinical trials have established that male circumcision could lower the risk of HIV acquisition in heterosexual men by as much as 62 percent. Sixty-two percent! So far, these studies have been limited to African populations that have been particularly hard-hit by AIDS-related casualties. In South Africa, a third of reproductive-aged women are infected. If you’re a 15-year-old living in that country today, there’s a 59 percent chance that you’ll die before reaching your 60th birthday; just 10 years ago, these odds were only 29 percent.Here’s how the clinical trials basically worked. Thousands of adult, HIV-negative, sexually active, uncircumcised men in Kenya, Uganda and South Africa agreed to be randomly assigned to a circumcision group or a no-circumcision group. Those randomly assigned to the circumcision group had their foreskins removed by medical professionals, were told to abstain from intercourse until their wounds healed (about three weeks—there may actually be a greater risk of HIV infection during this period, so this is vital), and then were instructed to return to the clinic at six-month intervals to test for the virus. The results were unequivocal: two years later, the circumcised males were significantly less likely than their uncircumcised peers to have contracted HIV. In fact, the researchers decided to end these clinical trials early for ethical reasons: with data so clearly showing the advantages of circumcision in an environment rife with the virus, it’s hard to justify a further wait-and-see approach for those men that had been randomly assigned to the no-circumcision group.For the Ugandan study, 22 of 2,387 circumcised men acquired HIV over the two-year period compared to 45 of 2,430 uncircumcised men who were infected during this time span. Extensive interview methods confirmed that the two groups did not differ in terms of their actual sexual behaviors, enabling the authors to conclude that the results were owed directly to the circumcision intervention. (For those data heads among you, P <>

The autoimmune polyglandular syndromes—a group of syndromes comprising a combination of endocrine and nonendocrine autoimmune diseases—differ in their component diseases and in the immunologic features of their pathogenesis. One of the three main syndromes, type 1 autoimmune polyglandular syndrome (APS-1), has a unique pathogenic mechanism owing to mutations in the autoimmune regulator (AIRE) gene, which results in the loss of central tolerance—a process by which developing T cells with potential reactivity for self-antigens are eliminated during early differentiation in the thymus. Patients with IPEX (immune dysfunction, polyendocrinopathy, enteropathy, X-linked) syndrome harbor mutations in the forkhead box P3 (FOXP3) gene in regulatory T cells, which leads to severe autoimmunity and immune deficiency. Although both of these disorders are rare, their well-defined mechanisms of disease provide a basis for the understanding of the more common condition, APS-2. In this syndrome, alleles of human leukocyte antigens (HLAs) determine the targeting of specific tissues by autoreactive T cells, which leads to organ-specific autoimmunity as a result of this loss of tolerance. Non-HLA genes also contribute to autoimmunity in APS-2 and, depending on the polymorphism, potentially predispose to a loss of tolerance or influence which organ is specifically targeted. This Review discusses the genetic basis of APS-1, APS-2 and IPEX syndrome, with an emphasis on the mechanisms of autoimmunity and presents currently available therapies to treat their underlying autoimmune disorders.

Key points

The type 1 autoimmune polyglandular syndrome results from mutations in the AIRE gene, which modulates transcription of peripheral self-antigens in the thymus presented by human leukocyte antigen (HLA) molecules to maturing T cellsThe type 2 autoimmune polyglandular syndrome is the most frequent autoimmune polyglandular syndrome, with underlying pathologies that develop years to decades apart in an affected individualPatients with Addison disease have a 50% risk of developing a second autoimmune disease during their lifetimeMutations in HLA genes, which encode the MHC (major histocompatibility complex) class II molecules expressed by antigen-presenting cells, contribute to the targeting of specific tissues by autoreactive T cellsNon-HLA genes contribute to the risk of autoimmune disease, as they may reduce the threshold of autoimmunity or influence the organs affected in type 2 autoimmune polyglandular syndromeIPEX (immune dysfunction, polyendocrinopathy, enteropathy, X-linked) syndrome results from mutations in the forkhead box protein P3 (FOXP3) gene, which is necessary for normal function of regulatory T cellsIntroductionImmunological tolerance is necessary for cells to distinguish foreign proteins and molecules from self-antigens. Central tolerance occurs in the thymus as T cells mature and can result in the development of effector T cells needed for host defense, regulatory T cells that suppress activation of the immune system and autoreactive T cells that react to proteins and tissues of the host. Central tolerance is composed of two selection processes; positive selection produces effector T cells and regulatory T cells that enter the circulation, whereas negative selection removes T cells with a high affinity for self-antigens. Peripheral tolerance occurs outside the thymus and acts as a safeguard to control any autoreactive T cells that survive negative selection in the thymus and reach the peripheral tissues. In healthy individuals, these autoreactive T cells are kept unresponsive by stimuli from regulatory T cells. Together, central and peripheral tolerance prevent the expansion and activation of autoreactive T cells and provide protection from autoimmune disease.The autoimmune polyglandular syndromes (APS) are a group of disorders characterized by the presence of a combination of multiple autoimmune disorders and, in some cases, immunodeficiency (Table 1). The syndromes include monogenic disorders such as APS-1 and IPEX (immune dysfunction, polyendocrinopathy, X-linked) syndrome, as well as complex genetic disorders, such as APS-2. Some of the underlying autoimmune disorders occur frequently in all syndromes, such as autoimmune thyroid disease and type 1A diabetes mellitus—the immune-mediated subtype of type 1 diabetes mellitus—whereas other components, for example, Addison disease and myasthenia gravis, are rare. Advances in the understanding of T cells and the process of tolerance to self-antigens have helped elucidate how certain individuals develop multiple autoimmune disorders.

Type 1 autoimmune polyglandular syndromeAPS-1, also called autoimmune polyendocrinopathy candidiasis ectodermal dystrophy (APECED), is a rare disorder, which generally manifests during infancy. The diagnosis is made when a child has at least two of the following pathologies: mucocutaneous candidiasis, hypoparathyroidism or Addison disease.1 Symptoms of mucocutaneous candidiasis—a recurring fungal infection that is limited to mucosal surfaces, skin and nails—usually manifest first. Hypotension or fatigue occurring as a result of Addison disease, or hypocalcemia owing to hypoparathyroidism, develop subsequently. APS-1 is associated with other autoimmune disorders (type 1A diabetes mellitus, vitiligo, alopecia, hepatitis, pernicious anemia and primary hypothyroidism) and, in one study,2 has also been linked to asplenism.PathogenesisOur understanding of tolerance has radically changed with the discovery of the autoimmune regulator (AIRE) gene and its role in the pathogenesis of APS-1. To enable elimination of the vast majority of autoreactive T cells, expression of peripheral, tissue-specific proteins not usually found in the thymus and their presentation to developing T cells is driven by the transcription factor AIRE.3, 4 This protein controls both intrathymic and extrathymic transcription of tissue-specific antigens. The panel of peripheral tissue antigens in lymph nodes and spleen is less diverse and different from those expressed in the thymus.5 In transgenic mice, deletion of the insulin gene specifically in medullary thymic epithelial cells that express Aire is sufficient to induce autoimmune diabetes mellitus and insulitis (the lymphocytic infiltration of pancreatic islets), despite normal expression of insulin in the β cells.6 Other peripheral antigens believed to be under the transcriptional control of AIRE include the enzyme steroid 21-hydroxylase, thyroid peroxidase, thyroglobulin and other autoantigens implicated in autoimmune disease.7APS-1 is a monogenic disorder which results from a mutation in AIRE. In total, over 40 mutations in the AIRE gene have been described,8 most of which are inherited in an autosomal recessive manner; however, a family with an autosomal dominant mutation has also been identified.9 Mutations in the AIRE gene result in decreased expression of the transcription factor and subsequently less presentation of self-antigens by medullary thymic epithelial cells and dendritic cells to developing T cells.10 Central tolerance to a number of self-antigens is lost, thereby inducing multiple autoimmune disorders as autoreactive T cells escape into the periphery. The specific tissues targeted by these autoreactive T cells is determined by human leukocyte antigen (HLA) alleles, which encode proteins on the surface of antigen-presenting cells that present antigens to T cells in the periphery when the immune system is activated.11Knockout of the Aire gene in animal models results in whole-body autoimmunity, although the phenotype is mild, with lymphocytic infiltration of the liver and atrophy of the adrenal and thyroid glands.12 The majority of mice also exhibit autoantibodies against proteins of the pancreas, adrenal glands, testes and liver.12 Studies in patients with isolated autoimmune disorders such as Addison disease, but without evidence of APS-1, have not found mutations in the AIRE gene.13TreatmentScreening for AIRE gene mutations is currently available. In addition, a previous study showed individuals with APS-1 have multiple anti-interferon antibodies, with interferon-ω-reactive autoantibodies present in 100% of patients.14 Assays for such autoantibodies can aid in the rapid diagnosis of APS-1.15 Hormone replacement is the mainstay of treatment for the endocrinopathies underlying APS-1. Mucocutaneous candidiasis must be treated aggressively and monitored for recurrence, as it can manifest anywhere along the gastrointestinal tract and, if left untreated, can lead to the development of epithelial cancers. If asplenism is identified, vaccinations against Streptococcus pneumoniae (pneumococcus) Neisseria meningitides (meningococcus) and Hemophilus influenzae should be administered.A high clinical suspicion for other autoimmune disease needs to be maintained when treating individuals with APS-1 and their first-degree relatives. A study by Cervato and colleagues16 found that 20 out of 25 relatives of individuals with APS-1 had heterozygous mutations in the AIRE gene. None of these relatives had APS-1 or any of the major autoimmune components (Addison disease, hypoparathyroidism or chronic candidiasis), but 40% had an underlying autoimmune disorder, including autoimmune thyroid disease, rheumatoid arthritis, polycythemia vera (a myeloproliferative disorder) and vasculitis.16 Patients with APS-1 must be followed at a center with experience in monitoring and caring for individuals with this condition. Siblings should be followed closely, and screening for anti-interferon-ω autoantibodies should be considered. Recommendations are to evaluate patients with APS-1 at 6-month intervals and screen for autoantibodies.17 If autoantibodies are present without the associated disease, functional testing is indicated. Patients with antibodies against steroid 21-hydroxylase should be tested annually, to evaluate and diagnose cortisol disorders, by stimulation testing with cosyntropin—a synthetic derivative of adrenocorticotropic hormone (ACTH)—unless symptoms or signs indicate that more frequent monitoring is needed. The presence of islet-cell autoantibodies (insulin, glutamic acid decarboxylase [GAD], islet antigen 2 [IA-2] and the zinc T8 transporter)18, 19 warrants home blood-glucose monitoring and glucose-tolerance testing to detect disease before overt clinical symptoms manifest.

Type 2 autoimmune polyglandular syndromeAPS-2, also known as Schmidt syndrome, is the most frequent autoimmune polyglandular syndrome. The defining component of APS-2 is Addison disease, which is found in conjunction with either autoimmune thyroid disease or type 1A diabetes mellitus. Women are typically affected at higher rates than men. Other diseases less frequently associated with APS-2 include celiac disease, vitiligo, pernicious anemia, myasthenia gravis, stiff man syndrome (a rare disease of unknown etiology characterized by progressive rigidity) and alopecia. First-degree relatives of individuals with APS-2 are at high risk of autoimmune disorders. A study of 10 families with APS-2 discovered that one in seven relatives had an undiagnosed autoimmune disease, the most frequent being autoimmune thyroid disease.20 Individual comorbidities can develop years to decades apart, which reinforces the necessity of a widespread knowledge of this syndrome, as early detection and treatment can prevent morbidity and mortality.

PathogenesisHLA genesAPS-2 is a complex genetic disorder governed by the HLA haplotype, which confers the risk of multiple autoimmune disorders. The HLA gene complex is located on chromosome 6 and divided into three classes (I, II and III). The class II alleles HLA-DQ and HLA-DR, and to a lesser extent HLA-DP, are the most important determinants of the underlying disorders of APS-2 and encode the major histocompatability complex (MHC) class II proteins located on antigen-presenting cells (macrophages, dendritic cells and B cells).21, 22, 23, 24

Much of our understanding of autoimmune disorders comes from the study of the component diseases in APS-2. Addison disease can be divided into stages of disease progression (Figure 1).25 Genetically predisposed individuals develop autoantibodies to the steroid 21-hydroxylase enzyme, gradually lose the ability to produce cortisol and eventually manifest with symptoms of adrenal insufficiency. Susceptibility to Addison disease is conferred through the genes encoding the MHC class II molecules, and there is a strong association with the HLA-DR3 haplotype.26, 27 The highest risk genotype, which occurs in 30% of patients with Addison disease, consists of the HLA-DR3/4-DQ2/8 haplotype.28, 29, 30 In this subgroup of patients, the HLA-DR4 subtype DRB1*0404 confers the highest risk of developing Addison disease, whereas the HLA-DR3 haplotype DQA1*0501,DQB1*0201 increases the risk for developing Addison disease in conjunction with type 1A diabetes mellitus and celiac disease. The HLA-DR4 haplotype of patients with all three of these diseases is associated with DQA1*0301,DQB1*0302. If a patient with type 1A diabetes mellitus expresses the DRB1*0404 allele and has steroid 21-hydroxylase autoantibodies, the risk of developing Addison disease increases 100-fold.31

The HLA class II molecules contribute to the specific disease development in APS-2; however, the contribution of non-HLA genes, such as the MHC class I chain-related A (MICA) 5.1 allele and the tyrosine-protein phosphatase non-receptor type 22 (PTPN22) gene, is emerging. Many genetic loci that have immunologic functions have been identified through genome-wide association studies, which analyze thousands of single-nucleotide polymorphisms in large populations to find alleles associated with a particular disease. These alleles can increase the risk of a certain disease (so-called high-risk alleles) or protect against it.

Insight into the pathogenesis of APS-2 comes from experimental animal models and human studies on genes that influence immune function. Two animal models of type 1A diabetes mellitus are the non-obese, diabetic (NOD) mouse and the Biobreeding (BB) rat. The NOD mouse is a model in which type 1A diabetes mellitus and sialitis—inflammation of a salivary gland or duct—develop spontaneously.32 As in humans, the NOD mouse carries a genetic predisposition towards the development of type 1A diabetes mellitus owing to specific alleles of genes within the MHC genomic region, which influence antigen presentation to T cells and the development of autoimmunity. However, other genes also mediate susceptibility for the development of type 1A diabetes mellitus in the NOD mouse, for example, the Iddm (insulin-dependent diabetes mellitus) genes located outside of the MHC region,33 of which 18 loci have been identified. Studies are underway to define the cellular mechanism by which these genes affect insulitis. Along with this genetic predisposition, an autoantigen is necessary for type 1A diabetes mellitus to develop. In NOD mice, one such self-epitope is a peptide of the insulin B chain (amino acids 9–23), which is recognized by autoreactive T cells.34, 35, 36 Depending on environmental triggers or the activation of the innate immune response, this autoantigenic peptide is presented to T cells and results in their activation. As the disease progresses, these activated T cells invade the pancreas and destroy β cells, which leads to insulin deficiency. Once β-cell destruction is initiated, other proteins become targets of the immune response including the β-cell specific islet glucose-related phosphatase (IGRP).37, 38, 39

The BB rat develops both type 1A diabetes mellitus and thyroiditis; HLA and non-HLA genes are required to determine the diabetic phenotype.40, 41 This strain has an autosomal recessive mutation in a non-HLA gene Iddm1 on chromosome 4, which results in a reduction of mature T cells in the blood.42, 43 Lymphopenia and the presence of a specific MHC allele (RT1U) are required for type 1A diabetes mellitus to develop. If lymphopenia occurs in combination with different MHC alleles, thyroiditis develops.44 The onset of autoimmunity is influenced by environmental factors that activate the innate immune system. When nonlymphopenic BB rats with the RT1U MHC allele were treated with the Kilham rat virus and polyinosinic-polycytidylic acid—an immunostimulant that activates toll-like receptor 3—all rats developed type 1A diabetes mellitus compared with only one third of the BB rats treated with virus alone.45

The lessons learned from these animal models are twofold. First, disease development is controlled by genes that encode the MHC class II molecules, along with non-HLA genes. Second, impaired regulation of the immune response is necessary for autoimmunity to develop. Failure to maintain tolerance results from a defective central tolerance and insufficient peripheral tolerance. Multiple autoimmune disorders develop when tolerance is lost to a number of self-antigens.

The MICA5.1 allele is associated with a genetic risk of Addison disease.46 Polymorphisms of the MICA gene differ in the number of triplicate GCT repeats in exon 5. The translated protein interacts with the NKG2-D type II integral membrane protein (NKG2D) receptor, which is important for thymic maturation of T cells.47 NKG2D can also regulate the priming of human naive CD8+ T cells.48 The allele is associated with the insertion of a base pair, which results in a premature stop codon and loss of the membrane binding region of the protein.49 This polymorphism is thought to result in the loss of central tolerance, with the escape of autoreactive T cells into the periphery.

Unlike MICA5.1, which predisposes to a deficiency in central tolerance, a group of long variable number of tandem nucleotide repeats (VNTRs) upstream of the insulin gene protects against the development of type 1A diabetes mellitus. The decreased risk of type 1A diabetes mellitus is associated with increased insulin expression in the thymus, and results in the deletion of autoreactive T cells in the thymus similar to that seen in individuals with a normal functioning AIRE gene.50 In other words, small differences in insulin action in the thymus have measurable effects on the development of autoimmunity and type 1 diabetes mellitus.

The PTPN22 gene influences immune function through the regulation of T-cell-receptor signaling. A single gain-of-function amino acid exchange (620R>W) in the gene product decreases T-cell-receptor signaling and increases the risk of many autoimmune disorders, including type 1A diabetes mellitus, Addison disease, Grave disease and rheumatoid arthritis.51 How a decrease in T-cell-receptor signaling enhances autoimmunity is unclear, but one could hypothesize that deficient negative selection of thymic autoreactive T cells might be involved.

Similar to PTPN22, the cytotoxic T lymphocyte protein 4 (CTLA4) acts as a negative regulator of T-cell activation. The CTLA4 antigen is located on the surface of T cells and interacts with CD28 on antigen-presenting cells as a negative signal to downregulate T cell activation, proliferation and differentiation.52 Depending on the polymorphism of the CTLA4 locus, risk of autoimmune disease is either increased or decreased, which equates to a stimulation or suppression of T cells, respectively. Polymorphisms in the 3'-promoter region and 5'-untranslated region predispose to type 1A diabetes mellitus,53 whereas a polymorphism in the 5'-promoter region protects against type 1A diabetes mellitus and autoimmune thyroid disease.54

TreatmentTreatment of APS-2 focuses on the identification and management of the underlying autoimmune conditions. Autoimmune thyroid disease is very common; levels of TSH in patients with type 1A diabetes mellitus and patients with Addison disease should, therefore, be determined at least once a year. Patients with Addison disease have a 50% lifetime risk of developing an additional autoimmune disease, and monitoring for other autoimmune disorders is crucial. These patients should be screened for autoantibodies against insulin, IA-2 and GAD for type 1A diabetes mellitus and tissue transglutaminase for celiac disease. The optimal screening interval is not defined; however, autoantibodies can develop at any age and repeated testing is, therefore, required. In addition, relatives of individuals with APS-2 need to be monitored closely.Immunotherapies are being used increasingly, particularly for the treatment of type 1A diabetes mellitus, in an attempt to halt the underlying autoimmune disease process. Therapies to modulate the immune response in type 1A diabetes mellitus aim to alter the processes that result in β-cell destruction and eventually lead to insulin dependence and consist of nonantigen-specific and antigen-specific agents. Nonantigen specific therapies target various components of the immune system and include those directed against T cells (anti-CD3 monoclonal antibodies,55, 56, 57 anti-thymocyte globulin58 and ciclosporin59) and B cells (anti-CD20 monoclonal antibodies).60, 61 Antigen-based therapies are believed to mediate immune tolerance to antigens that lead to autoimmunity of β cells. These therapies include vaccines with GAD,62 the B chain of insulin and other insulin-like peptides. Many of these therapies have reversed hyperglycemia in the NOD mouse, and several therapies show promise in humans.63

IPEX syndromeThe rare IPEX syndrome is caused by mutations in the forkhead box P3 (FOXP3) gene, which results in the absence or dysfunction of regulatory T cells.64 Clinically, this syndrome presents during the first few months of life with enteropathy, dermatitis, failure to thrive and multiple endocrinopathies. Affected neonates show overwhelming autoimmunity, which includes type 1A diabetes mellitus that can develop as early as 2 days after birth.PathogenesisTo date, 20 mutations in the FOXP3 gene have been identified in patients with IPEX syndrome.65, 66 Most of these mutations occur in the forkhead (winged-helix) domain and leucine zipper region of the gene product and prevent DNA binding. The inability of the transcription factor FOXP3 to bind DNA in regulatory T cells impairs immune suppressor function and leads to overwhelming autoimmunity. The scurfy mouse harbors a naturally occurring mutation in the gene that is homologous to the human FOXP3 gene and develops a disease very similar to IPEX syndrome, which enables the understanding of the disease pathogenesis and provides a model to evaluate treatment modalities.67, 68 Neonatal thymectomy in male scurfy mice ameliorates the disease and increases lifespan. Conversely, transfer of peripheral CD4+ T cells, but not CD8+ T cells, from mutant mice to wild type mice induces the disease in healthy animals.69 Peripheral CD4+ T cells from scurfy mice seem to be hyperresponsive to antigens and have a decreased requirement for co-stimulation with CD28,70 a molecule on T cells required for their activation. The inability of CD4+CD25+ regulatory T cells to regulate the immune response results in the IPEX syndrome.

TreatmentChildren affected with IPEX syndrome usually die in the first 2 years of life owing to a failure to thrive, malabsorption of nutrients or from infections that result from immunosuppressive therapy. Supportive care and treatment of underlying disorders is necessary and restoration of normal regulatory T-cell function can improve the symptoms of IPEX syndrome. Immunosuppressive medications (high-dose glucocorticoids, tacrolimus, ciclosporin, methotrexate, sirolimus, infliximab and rituximab) have been evaluated in case reports or small case series;71, 72, 73, 74, 75 however, toxic effects and complications arising from infections limit their use. In mice, the disease does not develop if the animals are mixed chimeras for scurfy and normal T cells,76 and the same seems to be true in humans with normal T cells that are able to suppress the abnormal T cells in a dominant fashion, as evidenced by bone-marrow transplantation reducing symptoms and prolonging survival77, 78, 79 of children with IPEX syndrome. Bone-marrow transplantation should be considered early on in the disease development to restore regulatory T-cell function, limit the autoimmune destruction to endocrine organs and possibly reduce the infectious complications from chronic immune suppression.

ConclusionsThe basic immunology underlying the pathogenesis of APS (Figure 2) has increased greatly over the past few years. The pathogenesis of APS-1 highlights the importance of intact central tolerance for the maintenance of self-tolerance. Mutations in the AIRE gene prevent expression and presentation of self-antigens to maturing T cells in the thymus. As a consequence, autoreactive T cells escape into the periphery and are able to induce tissue destruction upon activation. The IPEX syndrome is extremely rare but emphasizes the importance of regulatory T cells in the development of autoimmune disease. By understanding the mechanisms of these rare disorders, insight can be gained into how isolated autoimmune disorders and APS-2 develop. In APS-2, both HLA genes and non-HLA genes contribute to the disease risk. Polymorphisms in HLA genes contribute to the targeting of specific tissues by T cells, whereas non-HLA genes either lower the threshold for autoimmunity to develop or determine the organ-specific nature of the underlying diseases. Other factors, such as the environment and activation of the innate immune system, may increase the likelihood of developing these syndromes. A widespread knowledge of the underlying pathologies and their mechanisms will hopefully aid early diagnosis and treatment and improve health outcomes for patients with APS.

domingo, 25 de abril de 2010

Practical GeneticsEuropean Journal of Human Genetics (2010) 18, 511–518; published online 4 November 2009Menkes diseaseZeynep Tümer1 and Lisbeth B Møller11Kennedy Centre, Glostrup, DenmarkCorrespondence: Professor Z Tümer, Kennedy Center, Gl. Landevej 7, Glostrup DK-2600, Denmark. Tel: +45 4326 0155; Fax: +45 4343 1130; E-mail: zet@kennedy.dkAbstractMenkes disease (MD) is a lethal multisystemic disorder of copper metabolism. Progressive neurodegeneration and connective tissue disturbances, together with the peculiar ‘kinky’ hair are the main manifestations. MD is inherited as an X-linked recessive trait, and as expected the vast majority of patients are males. MD occurs due to mutations in the ATP7A gene and the vast majority of ATP7A mutations are intragenic mutations or partial gene deletions. ATP7A is an energy dependent transmembrane protein, which is involved in the delivery of copper to the secreted copper enzymes and in the export of surplus copper from cells. Severely affected MD patients die usually before the third year of life. A cure for the disease does not exist, but very early copper-histidine treatment may correct some of the neurological symptoms.

Keywords: Menkes disease; ATP7A; copperIn brief•Menkes disease (MD) is a lethal multisystemic disorder of copper metabolism.•Progressive neurodegeneration and connective tissue disturbances, together with the peculiar ‘kinky’ hair, are the main manifestations.•MD is inherited as an X-linked recessive trait, and as expected the vast majority of patients are males.•MD occurs due to mutations in the ATP7A gene.•The vast majority of ATP7A mutations are intragenic mutations or partial gene deletions.•ATP7A is an energy-dependent, transmembrane protein, which is involved in the delivery of copper to the secreted copper enzymes and in the export of surplus copper from cells.•Severely affected MD patients die usually before the third year of life. A cure for the disease does not exist, but very early copper–histidine treatment may correct some of the neurological symptoms.Top of pageIntroductionMenkes disease (MD) is an X-linked multisystemic lethal disorder of copper metabolism. Patients usually exhibit a severe clinical course, with death in early childhood, but variable forms exist and occipital horn syndrome (OHS) is the mildest form. The defective gene in MD (ATP7A) is predicted to encode ATP7A, which is involved in the delivery of copper to the secreted copper enzymes and in the export of surplus copper from cells.Normal and abnormal copper metabolism in human and other organisms has been the focus of extensive research, and tremendous knowledge has been accumulated on this subject. In this paper we will give a general view of MD and copper metabolism. Due to space restriction not all the original publications have been cited in this review, and relevant references can be found in two book chapters.1, 2Clinical synopsisMD shows considerable variability in its severity. Classical MD is the most severe form, while OHS is the mildest recognized form (reviewed by Tümer and Horn 2004, 20021, 2).Classical MDProgressive neurodegeneration and marked connective tissue dysfunction characterize the clinical picture of the most common severe form of MD, and death typically occurs before the third year of life (Figure 1).Pregnancy is usually uncomplicated. There may be premature labor and delivery, but most male patients are born at term with appropriate birth measurements. Cephalohematomas and spontaneous fractures are occasionally observed at birth. In the early neonatal period, patients may present with prolonged jaundice, hypothermia, hypoglycemia and feeding difficulties. Pectus excavatum and umbilical and inguinal hernias have also been reported. The first sign of MD may be unusual sparse and lusterless scalp hair that becomes tangled on the top of the head at the age of 1–2 months (Figure 2). At this time the appearance may be described as being odd, with pale skin, frontal or occipital bossing, micrognatia, pudgy cheeks, and a rather expressionless appearance. However, these changes are often too subtle to attract attention. Initial psychomotor development is usually unremarkable, with normal babbling and smiling up to about 2–4 months of age. The baby then ceases to develop further and gradually loses some of the previously developed skills. The developmental regression becomes obvious around 5–6 months of age. Most patients develop therapy-resistant seizures from about 2 to 3 months of age. Additional symptoms are failure to thrive, poor eating, vomiting, and diarrhea. Muscular tone is often decreased in early life, but is later replaced by spasticity and weakness of the extremities. As the motor dysfunction progresses, spontaneous movements become limited and drowsiness and lethargy emerge. The patients are typically diagnosed at 3–6 months of age, often due to the abnormal hair that is a striking feature of the disease. The hypopigmented or depigmented hair resembles and feels like steel wool; it is lusterless and friable, especially in the areas of the scalp subjected to friction.

Figure 2.Abnormal hair in a patient with classical MD. (a) Stubby appearance of depigmented scalp hair. (b) Hair microscopy ( × 100) of twisted hair shaft (pili torti) (below) and a normal hair strand (above) (courtesy of E Reske-Nielsen, Glostrup University Hospital, Denmark).Vascular, urogenital, and skeletal abnormalities are numerous. Patients have skeletal changes, including pectus excavatum or pectus carrinatum, and spontaneous fractures due to generalized osteoporosis. The joints are hyperextensive, and loose and dry skin may be observed very early. Thick, scaly seborrheic dermatitis is also a frequent feature. Routine ophthalmoscopy is usually normal, but in later stages patients frequently fail to follow visual stimulus.

Late manifestations of the disease are blindness, subdural hematoma, and respiratory failure. Most patients die within the third year of life due to infection, vascular complications (such as sudden and massive cerebral hemorrhage due to vascular rupture), or from the neurological degeneration itself.

The OHSOHS is the mildest recognized form of MD, and its principal clinical features are related to connective tissue. The main distinction between OHS and the other forms of MD is the radiographic observation of characteristic occipital horns (Figure 3). These are symmetric exostoses protruding from the occipital bone and pointing down.

Figure 3.Lateral skull radiograph of a 23-year-old OHS patient. The occipital exostoses (arrow) are not present at birth and become prominent by age (courtesy of I Kaitila, Helsinki University Hospital, Finland).Pregnancy is usually normal. The skin may appear wrinkled and loose at birth, and umbilical or inguinal hernias may be present. Within days, hypothermia, jaundice, hypotonia, and feeding problems may develop. Clinical problems become gradually obvious, and the first signs that bring the child to medical attention may be intractable diarrhea or recurrent urinary tract infections. In spite of these problems, diagnosis of OHS is usually made only around 5–10 years of age.Motor development is delayed due to muscular hypotonia and is associated with unusual clumsiness. Height is usually normal, while mild disproportion with long trunk, narrow chest and shoulders, thoracolumbar kyphosis or scoliosis, and pectus deformity are common. The joints are hypermobile. Elbow mobility is restricted and there is a tendency toward dislocation of the elbows. Facial appearance gradually becomes distinctive. Unusual features include long, thin face, often with a high forehead, down-slanting eyes, hooked or prominent nose, long philtrum, high arched palate, and prominent large ears. The extent of skin laxity is variable and may increase with age, resulting in droopy wrinkles around the trunk. Hair is usually not conspicuously abnormal, although some patients may have lusterless and unusually coarse hair. Recurrence of the inguinal hernia is common. Vascular anomalies, such as varicose veins, are common, and arterial aneurysms have also been described. A particular problem is orthostatic hypotension. The intellectual capacity is described as low to borderline normal. Pubertal development is normal.The clinical course is characterized by chronic diarrhea, bladder diverticulae with recurrent urinary tract infections and occasional spontaneous bladder ruptures, orthostatic syncope, and joint instability in the inferior extremities and limitations at the elbows. Some patients require surgery for severe progressive thoracolumbar kyphosis, spontaneous retinal ablation, or mitral valve insufficiency. Life expectancy in OHS is variable, although substantially longer than that in MD. There are adult patients up to 50 years who have maternal male relatives dying in early childhood (I Kaitila, personal communication).Intermediate phenotypesA number of MD patients with milder symptoms and later onset have been described. However these intermediate forms are not well categorized and different descriptions such as mild, moderate, or long surviving MD have been used.Normal and abnormal copper metabolismCopper is the third most abundant trace element in the body, after iron and zinc, and is required for normal function of several copper enzymes participating in important metabolic processes (Table 1). Copper is involved in cellular respiration (cytochrome-c oxidase (COX)); neurotransmitter biosynthesis (dopamine β-hydroxylase); maturation of peptide hormones (peptidyl α-amidating enzyme); free-radical scavenging (superoxide dismutase); cross-linking of elastin, collagen (lysyl oxidase) and keratin (sulfhydryl oxidase); melanin production (tyrosinase); and iron homeostasis (ceruloplasmin and hephaestin). Copper has further been implicated in myelination in regulation of the circadian rhythm, and may also be necessary for coagulation and angiogenesis (reviewed by Tümer and Horn 2004, 20021, 2).Table 1 - Mammalian copper enzymes and their suggestive relationship between MD symptoms.Full tableAlthough essential, owing to its chemical properties, the same metal may be highly toxic. Copper can exist in two oxidation states, Cu(I) and Cu(II), and reversible interchange between these two states is the basis of the enzymatic reactions. The same property, however, can result in the production of free radicals, which have detrimental effects on cellular components. Fine regulation of copper homeostasis is, therefore, vitally important for all living organisms.Cellular copper metabolismCopper uptake across the plasma membrane is likely to use an energy-independent membrane transporter (CTR1) (Figure 4).3 In the cytoplasma copper is bound to small proteins such as metallothionein4 and glutathione,5 or copper-specific chaperones, and thereby the cell is protected from the toxic effects of the free ion. The three known copper-chaperons, CCS, ATOX1, and COX17, bind the copper ion and guide it to different cellular locations, securing efficient delivery of the metal to the enzymes. CCS targets copper to superoxide dismutase, which resides in the cytosol or in mitochondria.6 COX17 guides copper to mitochondria to be incorporated into COX, where other proteins involved in COX copper metallation (such as COX11, SCO1 and SCO2) also reside (reviewed by Turski and Thiele7). ATOX1 guides copper to trans-Golgi network (TGN), where it is incorporated into copper-requiring enzymes synthesized in the secretory pathway.8Figure 4.Schematic illustration of cellular copper transport. Copper is taken up across the plasma membrane by the copper uptake transporter (CTR1) as cuprous ions (CuI). Within the cytoplasma, the copper is found attached to glutathione (GSH), metallothionein (MT), or copper chaperons, which deliver copper to enzymes and compartments. COX17 is the copper chaperone for COX, CCS is the copper chaperone for the cytoplasmic superoxide dismutase (SOD1), and HAH1 is the copper chaperone for ATP7A, which delivers copper to peptidyl-α-amidating enzyme (PAM), dopamine β-hydroxylase (DBH), tyrosinase (TYR), lysyl oxidase (LOX), and extracellular SOD3. ATP7A is also responsible for copper export from cells. At low copper concentrations, the localization of the protein is at the TGN, but at high copper concentrations it will be relocated to the plasma membrane. In the liver the role of ATP7A is performed by ATP7B.In the TGN two homologous membrane-bound, copper-specific ATPases, ATP7A and ATP7B (defective in MD and Wilson disease, respectively), transfer copper across the membrane into the lumen of the TGN, where it is delivered to secreted enzymes. ATP7A is expressed in almost every organ except the liver where ATP7B is predominantly expressed. In concordance with this, copper is incorporated in ceruloplasmin by ATP7B in hepatocytes, while ATP7A is in charge in most other cell types in transporting copper to tissue-specific enzymes. This also reflects why MD is a systemic disorder, while in Wilson disease, mainly the liver is affected.ATP7A (and ATP7B) has a dual role in the cell: apart from copper-loading of enzymes in the secretory pathway, it is responsible for ATP-driven efflux of copper from the cells.9 Under normal physiological copper concentrations ATP7A is localized to TGN, transporting copper into the lumen to the copper-dependent enzymes. Under increased copper concentrations ATP7A is translocated to the vesicles10 or to the plasma membrane.11Whole-body copper metabolismIn mammals the essential source for copper is the diet and for humans the average intake is about 1 mg/day. The dietary copper is absorbed from the intestinal lumen across the mucosal barrier into the interstitial fluid, and to portal blood. The non-specific metal transporters, DMT1, ATP7A, and CTR1, are involved in this multistage process.12, 13From the blood the copper is mainly transported to the liver and in lesser amounts to the kidney and other tissues including brain. In the liver, which is the central organ of copper storage and homeostasis, the copper is either secreted to the blood bound to ceruloplasmin or excreted to the bile. Both processes are controlled by ATP7B; defective ATP7B will, thus, lead to increased amounts of copper in the liver and other organs as observed in Wilson disease patients. The main excretory route of copper is bile and urinary loss is negligible.Free copper ions are virtually non-existing in living organisms. The concentration of free copper ions has been estimated to be in the order of 10−13 in the human blood14 where the metal is mainly bound to ceruloplasmin, albumin, and histidine. Ceruloplasmin is the major copper-containing protein component in serum, but it is disputable whether it has a role in copper metabolism.15 Albumin-bound copper is in equilibrium with amino-acid-bound copper and these two forms probably constitute a buffer system that secures the availability of sufficient copper to tissues as well as protecting against copper toxicity.The copper is transported to the brain across the blood–brain barrier at the cerebral endothelium and the blood–cerebrospinal fluid barrier at the choroid plexus. The details of copper transport to the brain are yet unknown. The copper transporters CTR1, ATP7A, and ATP7B are all expressed profoundly in brain barrier fractions, indicating a possible role of these transporters in brain copper uptake.16 In the brain ATP7A is involved in normal functioning of copper-dependent enzymes. Expression of ATP7A in the brain is developmentally regulated and it is high in the early postnatal period.17Copper homeostasis in MDElimination of copper from cells is the basic disturbance in MD, and almost all the tissues except for liver and brain will accumulate copper to abnormal levels., Although high, the copper level does not reach a toxic state in MD. This is partly due to an already diminished intestinal copper absorption, because of defective copper export from the mucosal epithelium, and partly due to the scavenger role of metallothionein.In the liver of MD patients, the low copper content is due to requirement of the metal in other tissues, rather than disturbed copper metabolism, as in the normal liver ATP7B, but not ATP7A, is the main copper transporter.The reason for the low copper content in the brain of MD patients is however different. The mammalian brain is one of the richest copper-containing organs in the body. Regulation of brain copper level is not well understood, but ATP7A must participate in this process, since MD leads to low copper levels in the brain. In MD patients, copper is likely trapped in both the blood–brain barrier and the blood–cerebrospinal fluid barrier, while the neurons and glial cells are deprived of copper.18 This also supports the role of ATP7A in brain copper uptake. Neuronal demyelination is also observed in MD patients due to ATP7A inactivation.19 Mediation of ATP7A-related copper release through NMDA-receptor activation suggests a new role of this protein in brain dysfunction other than through deprivation of copper-dependent enzymes.20 It is, thus, likely that seizures and neuronal degeneration observed in MD patients may also be related to a disturbed neuronal transmission through impaired function of NMDA receptors.20Structure of the ATP7A proteinATP7A (and ATP7B) is a member of a large family of P-type ATPases that are energy-utilizing membrane proteins functioning as cation pumps (Figure 5) (reviewed by Lutsenko et al22). They are called ‘P-type’ ATPases, as they form a phosphorylated intermediate during the transport of cations across a membrane. The super-family of P-type ATPases also includes the Na+/K+ and H+/K+ pumps, as well as plasma membrane and sarcoplasmatic reticulum Ca2+ pumps.21Figure 5.Schematic 3D protein structure of ATP7A with the functionally important domains. At the N-terminal ATP7A has six MBDs with the copper-binding motifs (CXXC). The protein is anchored to the membrane with the transmembrane domains (TMDs) and within TMD6 resides the CPC motif, which is assumed to play a direct role in copper translocation. The activation domain (A) with the invariant TGE residues, the nucleotide-binding domain (N), and the phosphorylation domain (P) with the invariant aspartate residue (D) are important domains for the catalytic activity of ATP7A.ATP7A (and ATP7B) transports copper across a membrane using the energy released by hydrolysis of ATP. This process (catalytic activity) involves domains specific for binding and hydrolysis of ATP, and is similar in all P-type ATPases. The domains involved in the catalytic cycle of the protein are the nucleotide-binding domain (N-domain), phosphorylation domain (P-domain), and activation domain (A-domain). Transport and translocation of copper furthermore requires special motifs and structures for recognition, binding, and translocation of the metal across a membrane. These motifs contain cysteine (C) residues, which play an important role in copper binding.At the N-terminus ATP7A has six metal-binding domains (MBD1–6) each with a consensus MTXCXXC motif. Copper binds to these domains in the reduced form, Cu(I). It is assumed that the two MBDs (MBD5 and MBD6) closest to the transmembrane domains are important for the functional activity of the protein, and at least one of these two sites is necessary for normal function of the protein. The first four metal-binding domains (MBD1–4) are thought to have a regulatory function. Interaction between ATP7A and the copper chaperone ATOX1 occurs through these domains.ATP7A is anchored to a membrane through eight hydrophobic transmembrane domains, which form a channel for copper translocation through the membrane, and the CPC motif within TMD6 is assumed to play a direct role in the transfer of copper.The catalytic activity of ATP7A is likely mediated through a coordinated action of the N-domain, the P-domain, and the A-domain. The N- and P-domains reside between TMD6 and TMD7. N-domain binds ATP and the γ-phosphate of ATP is then transferred to the invariant aspartate residue (D) in the DKTG motif, which resides in the P-domain. This results in the formation of a transient phosphorylated intermediate. Following translocation of the copper through the membrane, the P-domain is dephosphorylated. The A-domain is located between TMD4 and TMD5, and includes the invariant TGE motif, where the glutamate residue (E) plays a key role in dephosphorylation of the phosphorylated intermediate.At the C-terminal ATP7A contains conserved di-leucine residues (LL), which is necessary for retrieval of the protein from the membranes (plasma membrane or vesicles).

Genetic basis of MDThe incidence of MD is calculated as 1 in 300 000 based on observations from a large population in five European countries,23, 24 and 1 in 360 000 in Japan.25 In Australia the incidence is reported to be much higher (1 in 50 000–100 000),26 but this might be due to a founder effect.

As expected the vast majority of MD patients are males, although a few female patients have also been reported. Most of the female patients have an X;autosome translocation, where the normal X-chromosome is preferentially inactivated.29 However, female patients with a point mutation and skewed inactivation of the normal X-chromosome have also been observed (unpublished data).

Chromosome abnormalities affecting ATP7A were detected in eight patients, one male33 and seven female patients. One of the female patients was mosaic for the Turner karyotype and the rest had X;autosome translocations (reviewed by Sirleto et al34). Approximately 25% of the ATP7A mutations (n=50) are gross deletions ranging in size from a single exon to deletion of the whole gene except for the first two exons.31 To date about 120 different intragenic mutations of ATP7A have been reported: missense (33%), nonsense (16%) and splice-site mutations (16%), and deletions/insertions/duplications (33%) (HGMD).28, 29, 32

About half of the point mutations (deletions/insertions/duplications and nonsense mutations) are truncating mutations, which are shown or predicted to result in a non-functional truncated protein. These truncating mutations are distributed almost equally throughout the gene, and they are predicted to have detrimental effects on the protein function. One interesting aspect of distribution of the mutations is that in the copper-binding domains of ATP7A encoded by exons 2–7, no missense mutations are observed. This suggests that variations in this region are more acceptable and do not necessarily lead to MD. Almost all the disease-causing missense mutations affect residues, which are within the regions conserved among ATPases. Several of the missense mutations leading to amino-acid substitutions have been investigated by functional studies for their effect on protein function (reviewed by de Bie et al35). The mutations may affect protein synthesis, stability of the protein, trafficking and localization, catalytic activity, and post-translational modifications. Missense mutations within the stalk regions or the transmembrane domains may lead to partially functional protein variants containing single-amino-acid substitution. In contrast, missense mutations in the domains important in catalysis (A- and P-domains) are poorly tolerated.32

Effect of the mutation on the phenotypeThere is no obvious correlation between the mutations and the clinical course of MD. This is underscored by the presence of inter- and even intra-familial phenotypic variability in MD/OHS patients carrying the same ATP7A mutation.36, 37In general patients with a milder phenotype (like OHS) have a higher proportion of mutations, which lead to a partially functional protein or result in reduced amounts of an otherwise normal protein.32 Splice-site mutations normally disturb the splicing process and lead to skipping of one or more exons. However, in some cases splice-site mutations do not lead to full disturbance of normal splicing and small amounts of normal transcript (and hence normal protein) can be produced. This has been observed in several OHS patients38, 39, 40 and in patients with a milder phenotype.41 Presence of partially functional, truncated protein variants missing only part of the C-terminus might also result in OHS or a mild Menkes phenotype.31, 42 Interestingly, in a patient with a mild phenotype we have detected deletion of exons 3–4, which resulted in a premature stop codon just at the beginning of exon 5, but translation was reinitiated from a downstream start codon.43 This truncated protein was partially functional with only two copper-binding domains (MBD 5–6), resulting in milder symptoms.Another mutational mechanism observed in ATP7A is the skipping of exons including the mutations.28 This may also explain why some patients may have milder phenotypes, as these mutations would produce in-frame deletions resulting in a partially functional protein. It is therefore necessary to investigate the effect of ATP7A mutations with functional analyses both at the transcript and protein levels, to be able to predict their effect on the clinical phenotype.In general gross gene deletions result in the severe classical form of MD, with death in early childhood. However, patients with gross deletions may also have long survival despite severe symptoms. In one patient who died at age 18 years, all the exons of ATP7A except for the first two exons were deleted.31

Animal modelsThe mottled mouse (mo), which has mutations in atp7a (the mouse orthologue of ATP7A), provides an animal model for MD. Mutations in the mottled locus are common and at least 35 different mutations lead to a wide range of neurological and connective tissue abnormalities. Most of the mottled mutations arise spontaneously and Atp7a mutations have been defined in 12 mottled mutants until now.44, 45, 46The mottled brindled (Mobr) and mottled macular (Moml) are the closest models to the classical form of MD. They both present with severe neurological impairment and hemizygous males die in the postnatal period (1–3 weeks). Atp7a mutations of both mottled alleles have been identified.47, 48, 49, 50 When Mobr is treated with copper injections within the first week of postnatal period the mice survive and do not develop neurological symptoms.51 Furthermore, transgenic expression of human ATP7A in Mobr could correct the phenotype even though copper defect was not completely corrected.52The mottled blotchy (Moblo) phenotype resembles OHS by showing predominantly connective tissue manifestations. Moblo has a splice-site mutation (IVS11+3A>C), which affects normal RNA splicing,39 but a normal transcript at a reduced level is also present, as is the case with the mutations of several OHS patients.Besides the mouse model, two zebrafish mutants, calamity and catastrophe, defective in the orthologue of ATP7A were identified.53, 54 Very recently the phenotypic alterations of two calamity mutant alleles have been corrected by antisense therapy.55

Diagnostic approachesInitial diagnosis of MD is suggested by clinical features (especially typical hair changes) and supported by demonstration of reduced levels of serum copper and ceruloplasmin (Figure 6). However, in the neonatal period these markers should be interpreted with caution, as their levels are low also in healthy newborns. In this period, plasma catecholamine analysis (ratio of DOPA to dihydroxyphenylglycol) indicative of dopamine β-hydroxylase deficiency may be the choice as a rapid diagnostic test.41Figure 6.Flow chart for diagnosis. The DOPA/DHPG ratio (ratio of dihydroxyphenylalanine to dihydroxyphenylglycol) is indicative of dopamine β-hydroxylase, which is a copper-dependent enzyme. Gross deletions were previously investigated with the Southern blot method, which is now replaced by MLPA (multliplex ligation-dependent probe amplification) method (LBM, unpublished data).Other laboratory investigations such as cystourethrography, arteriography, computed tomography, magnetic resonance imaging, and radiography are useful in detecting different clinical features of MD.Radiographs of patients with classical MD show a number of specific abnormalities that are reminiscent of acquired copper deficiency and scurvy. These changes include generalized osteoporosis, metaphyseal flaring and spurs in the long bones, diaphyseal periosteal reaction and thickening, and Wormian bones in the cranial sutures. Rib fracture due to osteoporosis is a common finding and may lead to misdiagnosis of battered child syndrome. In OHS patients occipital horns are characteristic radiographic findings (Figure 3). These protrusions may be found around 1–2 years of age, but are usually detected only around 5–10 years of age. They continue to grow up to early adulthood.Light microscopy of hair shows individual hairs that are twisted about their own axes (pili torti), with varying shaft diameters (monilethrix), and fragmentation at regular intervals (trichorrhexis nodosa) (Figure 2).A definitive biochemical test for MD exists and is based on intracellular accumulation of copper due to impaired efflux. Accumulation is evaluated in cultured cells, mainly fibroblasts, by measuring radioactive copper (64Cu) retention after a 20-h pulse, and impaired efflux is directly determined after a 24-h pulse–chase period. However, these analyses demand expertise and are performed only in a few specialized centers around the world.56

The ultimate diagnostic proof of MD is the demonstration of the molecular defect in ATP7A. However, because of the large size of the gene and the variety of the mutations observed in different families, detection of the genetic defect in a given family may take time.

Carrier identification and prenatal diagnosisIn at-risk families only male fetuses need to be evaluated, and rapid sex determination can be made using Y-chromosome-specific DNA sequences.

Carrier determination by measuring radioactive copper accumulation in cultured fibroblasts is not reliable due to random X-inactivation, and mutation analysis should be performed. In informative families, the intragenic polymorphic markers may also be used for carrier detection.

In at-risk pregnancies where the mutation of the family is unknown, biochemical analysis remains a possibility as identification of the genetic defect may be challenging in limited time. In the first trimester the total copper content in chorionic villi can be measured directly by sensitive and accurate methods like neutron activation analysis and atomic absorption, and in the second trimester copper accumulation is measured in cultured amniotic fluid cells. Although there are potential pitfalls for these analyses, they have been performed routinely at the Kennedy Center in Denmark since 1975.56

TreatmentMD is a progressive disorder leading to death in early childhood in its severe forms, although some patients survive above 5 years of age. Treatment in major cases is mainly symptomatic and clinical reports suggest that care is an important factor in enhancing survival. However, careful medical care, and possibly copper administration, may extend life span up to 13 years or even more. A number of severely affected MD patients with long survival have been reported.The objective of a specific treatment for MD is to provide extra copper to the tissues and copper-dependent enzymes. Oral administration of copper is ineffective as copper is trapped in the intestines; it should be supplemented parenterally or subcutaneously. Among the available copper compounds, copper histidine has proved to be the most effective.57, 58 Positive outcome of copper–histidine supplementation is dependent on early initiation and presence of at least partially functional ATP7A.Early, parenteral copper–histidine supplementation may modify disease progression substantially, and the long-term clinical outcome of four early-treated MD cases has been reviewed.58 All these patients clearly exhibited a milder neurological course without seizures, mild-to-moderate ataxia, and near-normal intellectual development. Some problems related to autonomic failure, such as postural hypotension and chronic diarrhea, persisted, and in one patient these symptoms could be corrected by L-DOPS. Copper–histidine treatment, however, could not prevent skeletal abnormalities and they have developed some features common to OHS patients. One of these patients is almost 30 years old now (manuscript in preparation).Early, subcutaneous copper–histidine treatment has also been described recently for 12 younger patients (the oldest patient is 8 years old), with good results.59 However, copper–histidine treatment cannot be accepted as a definitive cure for MD, despite some reported successful outcomes.References1.Tümer Z, Horn N: Menkes disease; in Roach ES, Miller VS (eds): Neurocutaneous Syndromes. Cambridge: Cambridge University Press, 2004, pp 222–233.2.Horn N, Tümer Z: Menkes disease and the occipital horn syndrome; in Royce PM, Steinmann B (eds): Connecitive Tissue and its Heritable Disorders: Molecular, Genetic, and Medical Aspects. New York: John Wiley and Sons Inc., 2002, 2nd edn 651–685.3.Lee J, Penea MMO, Nose Y, Thiele DJ: Biochemical characterization of the human copper transporter Ctr1. J Biol Chem 2002; 25: 40253–40259. Article ChemPort4.Formigari A, Irato P, Santon A: Zinc, antioxidant systems and metallothionein in metal mediated-apoptosis: biochemical and cytochemical aspects. Comp Biochem Physiol C Toxicol Pharmacol 2007; 146: 443–459. Article PubMed ChemPort5.Speisky H, Gómez M, Burgos-Bravo F et al: Generation of superoxide radicals by copper-glutathione complexes: redox-consequences associated with their interaction with reduced glutathione. Bioorg Med Chem 2009; 17: 1803–1810. Article PubMed ChemPort